Characterisation of the secondary protein structure within the lipidic extrudate

FTIR-spectroscopy has been suggested to analyse the secondary structure of proteins embedded within controlled release devices [75, 228, 256]. This method inheres the benefit that extraction of the protein is not necessary as the protein structure is directly studied within the delivery system.

FTIR-spectra of solid protein can be obtained for instance with the ATR-technique or with the transmission-technique using infrared-transparent salts. The main advantage of the ATR-technique is that it obviates time consuming sample preparation. In brief, the basis of ATR-spectroscopy is a beam of infrared radiation entering an optically transparent crystal of a higher refraction index than the sample. At the crystal-sample

interface the beam is totally reflected and consequently an evanescent wave penetrates the sample located at the crystal surface at the reflections point. In comparison, the IR-beam passes directly through the sample when the KBr transmission method is applied to collect the spectra [230].

In a pilot survey the potential of ATR- and transmission-technique to characterise the secondary structure of IFN-α embedded in a lipidic powder formulation was investigated. In accordance to the extrudate formulations presented in Chapter V.2.1 a lipid mixture based on H12 and tristearin in a ratio of 1 to 4 was blended with 10 % IFN-α and with 10 % PEG. As illustrated in Figure 90 the spectra recorded by the ATR-technique revealed a protein signal in the amid l/amid II region. However, better signal to noise ratio was obtained in the transmission mode. Thus, it was decided to use this technique for further investigations.

1.19 1.195 1.2 1.205 1.21

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1800 2000

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absorbance

A

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placebo blend protein blend

B

Figure 90: ATR-spectrum (A) and KBr pellet transmission spectrum (B) of the physically mixed lipid, PEG and protein formulation

As many proteins revealed spectral changes upon lyophilisation [33, 230] first the KBr transmission spectra of lyophilised IFN-α were recorded. The second derivative transmission spectra of IFN-α lyophilised with HP-β-CD and that of the protein in solution are shown in Figure 91.

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1640 1660

1680 1700

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Figure 91: Vector-normalised second-derivative spectra of IFN-α.

The solid line represents the second-derivative spectra of lyophilised IFN-α obtained with the KBr pellet method. The dashed line shows the second-derivative spectra native IFN-α in solution.

Upon lyophilisation the dominant band ascribed to the helical structure was shifted from 1654 cm^-1 to 1656 cm^-1. A shift of the band positions upon lyophilisation may indicate variations in the order of the α-helical structure [175]. Furthermore, a broader amid I band in the KBr spectra was observed and a new band at 1615 cm^-1 arose.

The occurrence of new bands at 1619 cm^-1 upon lyophilisation was related to intermolecular β-sheet formation [229].

Though Carpenter and co-workers showed that the extent of spectral changes occurring upon lyophilisation correlated with the storage stability [33], there is still a debate about the reasons for these structural changes. For instance, Van de Weert et al. [229] figured out that both structural changes as well as the removal of water might induce differences in the absorption characteristics of the amid bond.

Within the scope of the present work the origin of the spectra differences was not further investigated. The spectra of lyophilised IFN-α should only serve as standard to evaluate the effects of the extrusion procedure on the secondary protein structure.

As shown before, the C=O stretch vibrations of the triglyceride matrix material occurred at 1737, at 1729, and at 1690 cm^-1 (see Figure 90). In particular the latter band overlapped with the amid I band of the protein. Hence, it was necessary to evaluate the possibility of background correction. These investigations were performed with IFN-α blended in a mortar with the lipidic matrix material to exclude potential changes of the protein structure. The obtained spectra of the lipid/protein powder blend (10 % IFN-α/HP-β-CD co-lyophilisate, 10 % PEG, 64 % tristearin, and

16 % H12) were corrected by the spectra of the respective placebo blend. Yang et al.

[256] suggested as criterion for successful background subtraction a flat baseline in the region at 1730 - 1710 cm^-1 (final protein spectra obtained after correction with PLGA background). However, it was not possible to fulfil this request with the lipidic/protein blend studied here (Figure 92).

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substraction factor 1.0 substraction factor 1.14 substraction factor 1.2

Figure 92: Effect of background subtraction on the KBr pellet transmission spectra of IFN-α embedded in a lipid/PEG blend

Variations of the subtraction factor either involved an oversubtraction or an undersubtraction of the lipid contributions, leading to negative or positive features in the spectrum between 1700 to 1800 cm^-1. The same problem was noted by van de Weert et al. [228]. In the cited work it was tried to correct the spectra of lysozyme embedded within PLGA microspheres by the spectra of the blank PLGA microspheres. The fact that the background could not completely cancelled out was explained by baseline slopes and distorted peak shapes especially in the carbonyl stretch vibration of PLGA due to the compression in KBr pellets. In addition, it was suggested that different absorption characteristics of protein–loaded microspheres restricted the subtraction of the spectrum of blank PLGA microspheres [228].

However, in accordance to the work of van de Weert et al. [228], the peak positions below ~1670 cm^-1 in the absorbance spectra as well as in the second derivative spectra were not markedly affected by the used subtraction factor (Figure 93).

Importantly, the obtained spectra were quite similar to that of lyophilised protein before blending with the lipid matrix material. Taking all together led to the conclusion

that the subtraction of the lipid background did not cause spectral artefacts in the amid I region below 1670 cm^-1 (Figure 93).

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substraction factor 1.0 substraction factor 1.14 substraction factor 1.2 lyophilised IFN-α

A

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B

Figure 93: Vector-normalised KBr-pellet-transmission spectra (A) and respective second derivatives (B) of IFN-α embedded in a lipid/PEG blend after background correction.

In Figure 94 the obtained second derivatives of IFN-α after extrusion with a H12/tristearin blend are illustrated. Compared to the physical blended protein no significant differences in the amid I region below ~1670 cm^-1 were detected.

The formation of intermolecular β-sheets would be detectable by the appearance of new bands at 1623-1641 cm^-1 and at 1674-1695 cm^-1 [230], but there were uncertainties in the latter band region with the described KBr transmission technique.

However, a structural rearrangement should be associated with a decrease in the α-helical content (compare Figure 54 (FTIR-spectra during thermal induced denaturation)). As shown in Figure 94 the band at 1653 cm^-1 was largely unaffected by the extrusion procedure. Thus, it is highly likely that extrusion of IFN-α embedded in a H12/tristearin blend comprising either 10 % or 20 % PEG did not induce important changes in secondary protein structure.

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wavenumber, cm^-1

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placebo blend lyophilised IFN-α

extrudate H12/tristearin 10% PEG extrudate H12/tristearin 20% PEG

Figure 94: Second derivative KBr-pellet-transmission spectra before extrusion and after extrusion with a lipidic blend based on H12 and tristearin.

In contrast, the second derivatives of IFN-α after extrusion with a Miglyol^®/tristearin blend revealed a number of differences compared to the spectra of the physical blend (Figure 95). The band arising from α-helical structures was shifted to 1653 cm^-1 with a decrease in the relative intensity. In addition, the absorption bands at 1635 cm^-1 and at 1619 cm^-1 gained in intensity. Based on the overall spectral perturbations, a modification of the secondary structure of IFN-α could be supposed upon extrusion with a Miglyol^®/tristearin blend.

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placebo blend lyophilised IFN-α

extrudate Migyol®/tristearin 10% PEG

Figure 95: Second derivative KBr-pellet-transmission spectra before extrusion and after extrusion with a lipidic blend based on Miglyol^® and tristearin.

In summary, SDS-PAGE analysis and FTIR-spectroscopy demonstrated that protein integrity was kept during extrusion at 40 °C with a lipid blend comprising H12 and tristearin. In contrast, SDS-PAGE analysis and FTIR-spectroscopy of IFN-α processed with the Miglyol^®/tristearin blend revealed the occurrence of higher-order aggregates and significant perturbations of the secondary protein structure even though extrusion was performed at room temperature.

Im Dokument Lipidic Implants for Pharmaceutical Proteins (Seite 173-179)